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Endocrinology Vol. 140, No. 8 3633-3642
Copyright © 1999 by The Endocrine Society

ARTICLES

Proparathyroid Hormone Processing by the Proprotein Convertase-7: Comparison with Furin and Assessment of Modulation of Parathyroid Convertase Messenger Ribonucleic Acid Levels by Calcium and 1,25-Dihydroxyvitamin D31

Lucie Canaff2, Hugh P. J. Bennett, Yu Hou, Nabil G. Seidah and Geoffrey N. Hendy

Departments of Medicine (L.C., H.P.J.B., Y.H., G.N.H.), Physiology (G.N.H.), and Human Genetics (G.N.H.), McGill University and Royal Victoria Hospital, Montréal, Québec H3A 1A1, Canada; and the J. A. DeSeve Laboratory of Biochemical Neuroendocrinology (N.G.S.), Clinical Research Institute of Montréal, Université de Montréal, Montréal, Québec H2W 1R7, Canada

Address all correspondence and requests for reprints to: Geoffrey N. Hendy, Ph.D., Calcium Research Laboratory, Room H4.67, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec H3A 1A1, Canada. E-mail: gnhendy{at}med.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic ß-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALL BIOSYNTHETIC enzymes are potential points of regulation, and the proprotein convertases (PCs) are no exception. For example, PCs that process proinsulin in the pancreatic ß-cell are regulated by blood glucose levels. Additionally, biosynthetic enzymes are targets for therapeutic intervention in, for example, disorders involving excessive hormone production. Therefore, understanding the roles of PCs in the processing of molecules such as proparathyroid hormone (proPTH) or the related gene family member proPTH-related protein will aid in the design and development of enzyme inhibitors of potential value for the treatment of hyperparathyroidism (1) and the hypercalcemia of malignancy syndrome (2). In the present study, we have examined the potential for involvement of PC7 in proPTH processing.

The messenger RNA (mRNA) for PTH, the major regulator of calcium homeostasis, encodes a pre- (or signal) sequence of 25 amino acids and a basic pro-peptide of 6 amino acids (3). After entry of the nascent peptide chain into the intracisternal space bounded by the endoplasmic reticulum, the pre-sequence is cleaved, proPTH is then transported to the trans-Golgi network (where the propeptide is removed), and the mature PTH polypeptide of 84 amino acids is packaged into secretory granules (4). Several mammalian subtilisin-like serine endoproteases have been described that process proproteins by cleaving at pairs of basic residues. These include furin [paired basic amino acid cleaving enzyme (PACE)] (5, 6), PC1(PC3) (7, 8, 9), PC2 (7, 10, 11), PACE4 (12), PC4 (13, 14), and PC5(PC6) (15, 16). Furin has a neutral pH optimum and functions in the trans-Golgi network, whereas PC1 and PC2 have more acidic pH optima and act predominantly within secretory granules. We previously assessed which of these enzymes could process proPTH to PTH (17). Cultured cell lines were coinfected with vaccinia virus (VV) constructs expressing either furin, PC1, or PC2 together with proPTH. PTH biosynthetic products were purified by reversed-phase (RP)-HPLC and identified by mass spectrometry. The coinfection studies revealed that furin was the most effective at processing proPTH to PTH. Colocalization studies, using Northern blot analysis and in situ hybridization, showed that furin is expressed in the parathyroid, whereas PC1 and PC2 are not (17). The parathyroid chief cell is therefore unusual in this respect, given that the vast majority of endocrine cells do express PC1 and/or PC2. Additionally, furin efficiently processed a 13-amino acid peptide spanning the prohormone cleavage site in proPTH (17), and that substitution of key amino acids within the site compromised its cleavage by furin (18). Therefore, on several grounds, furin is a strong candidate to be the enzyme responsible for the physiological processing of proPTH to PTH.

The most recently characterized member of the PC family is PC7 (19) [also known as LPC (20), PC8 (21), or SPC7 (22)]. PC7 has a pH optimum, Ca2+ dependence, and cleavage specificity largely similar to furin and is also membrane-anchored (23). Also, like furin, PC7 has a very widespread tissue distribution (19, 20), which suggests that it is involved predominantly in the processing of precursors within the constitutive secretory pathway. At the time of our initial study of PCs and proPTH (17), PC7 had not been identified. In the present study, we investigated whether PC7 might be involved in proPTH processing. Here, we examined using the coexpression approach, the relative abilities of furin, and PC7 to correctly process proPTH to PTH. This revealed that PC7, as well as furin, can appropriately cleave proPTH in cells having a regulated secretory pathway. Colocalization studies showed that PC7 is expressed at low levels in the parathyroid gland. Therefore, PC7 (like furin) has the potential to be involved in the physiological processing of proPTH to PTH.

The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D levels. For example, calcium negatively regulates PTH biosynthesis (24) and secretion (25), and 1,25(OH)2D3 also negatively regulates PTH biosynthesis (26). We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems, in which regulation of PTH mRNA levels were clearly demonstrated, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin mRNA levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vaccinia virus constructs
The purified VV constructs used were as follows: The VV recombinant of human (h) proPTH (VV:hPTH) was prepared using a full-length hpreproPTH complementary DNA (cDNA) (27). The recombinant VV:hfurin was prepared using hfurin cDNA (28) (kindly provided by Dr. A. Rehemtulla, Genetics Institute, Cambridge, MA), subcloned into the pVV3 transfer vector (29). Likewise, VV:rPC7 was prepared from a full-length cDNA of rPC7 (19), and VV:5'KrPC7 was generated by PCR modification of the native translation start site sequence CTGATGC to GTGATGG [a consensus Kozak protein translation start site (30)]. The VV recombinant of rat dynorphin (VV:rdyn) was prepared as described previously (31).

VV infections
Rat pituitary tumor GH4C1 cells, which have a regulated secretory pathway in addition to the constitutive pathway, were infected with a mixture of VV:hPTH and either VV:rdyn (control), VV:hfurin, VV:rPC7, or VV:rPC75'K as described previously (32). After the infection period, the inoculum was replaced with DMEM, and cells were incubated for 17 h at 37 C. The cells were then incubated in DMEM containing 0.01% BSA for 4 h, after which cells were harvested for further analysis.

RP-HPLC of culture media and cell extracts
Both culture media and cell extracts were separately subjected to RP-HPLC (33) using a C18 µ-Bondapak column (Waters, Milford, MA), which was eluted over 1 h with a linear gradient of 16–56% aqueous acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid (CF3COOH) at a flow rate of 1.5 ml/min. Under these conditions, PTH and all known parathyroid cell-derived PTH fragments are eluted from the column (34). The presence and nature of immunoreactive PTH moieties in column fractions were determined by Western blot analysis and mass spectrometry, respectively.

Western blot analysis
Chromatography fractions were electrophoresed through tricine-SDS-polyacrylamide gels containing 16.5% (wt/vol) acrylamide, 3% (wt/vol) bisacrylamide, designed to optimally resolve proteins or polypeptides below a molecular mass of 20 kDa (35). The resolved proteins were blotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada). Membranes were rinsed in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (TBST), blocked with 5% dried milk powder in TBST for 1–2 h and incubated with specific antibodies. The specific antisera used were G150 raised against hPTH-(1–84) (17) and R1249 raised in a rabbit against a synthetic tridecapeptide, hproPTH(-6-+7), corresponding to amino acids -6 to +7 of the hproPTH molecule (with +1 designating the first amino acid of the mature 84-amino acid molecule), as a multiple antigen peptide (prepared by solid-phase chemistry in the Peptide Synthesis Facility of the Sheldon Biotechnology Centre of McGill University). Antibody-antigen complexes were detected by chemiluminescence using the LumiGlo chemiluminescent substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Extracts of Escherichia coli (E. coli) expressing recombinant hproPTH (36) and hPTH (37) were used as controls.

Mass spectrometry
Chromatography fractions were analyzed for their peptide content by delayed extraction matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using a Voyager-DE mass spectrometer (PE Biosystems, Framingham, MA) located at the Sheldon Biotechnology Centre of McGill University. One-microliter aliquots of each HPLC column fraction were mixed with an equal volume of a saturated solution of matrix ({alpha}-cyano-4-hydroxycinnamic acid, Aldrich Chemical Co., Inc., Milwaukee, WI) in 40% aqueous acetonitrile containing 0.1% (vol/vol) CF3COOH and were allowed to air dry on the 100-sample plate. Masses were scanned in the range 500–12,000 and were recorded as a plot of signal intensity vs. the mass-to-charge ratio. Because MALDI-TOF/MS predominantly measures singly charged species, especially in the low-mass ranges, spectra can be interpreted as representing the molecular weight plus one (i.e. the single proton incorporated during the process of ionization). Thus, the mass-to-charge ratio values correspond to the peptide mass plus one over the charge, which in this case is also one. Mass spectra of column fractions were inspected for signature signals close to that corresponding to the predicted values for hPTH and hproPTH (i.e. 9,425 and 10,152, respectively). Note that these values represent the predicted masses for the two peptides using average isotopic values for each amino acid, plus one mass unit corresponding to the proton incorporated during ionization. Mass estimates in the 10,000 molecular weight range were expected to be within 4 mass units of theoretical values according to specifications of the manufacturer of the mass spectrometer.

Preparation of parathyroid cells
Bovine parathyroid cells were collected at a local slaughterhouse and were transported to the laboratory in ice-cold MEM containing antibiotics, 1 mM CaCl2, and 0.5 mM MgCl2. Dispersed cells were prepared as described previously (38, 39). The parathyroid glands were rinsed briefly in 70% ethanol and placed in fresh medium. They were trimmed of excess fat, minced with scissors, and separated from the medium by centrifugation (500 rpm, 5 min), then suspended in 50 ml digestion medium consisting of MEM containing 150 mg collagenase (Worthington Biochemical Corp., Freehold, NJ) and 2 mg deoxyribonuclease (Roche Molecular Biochemicals, Laval, Québec, Canada), 1 mM CaCl2, and 0.5 mM MgCl2. The mixture was incubated at 37 C for 3 h, with shaking (80 cycles/min) and vigorous pipetting every 20 min to disperse the cells. Cells were filtered through a 150-µm mesh and washed three times with PBS.

Parathyroid cell cultures
Washed cells were suspended in MEM, 10 mM HEPES, antibiotics, 5% FCS, 0.5 mM MgCl2, and 1 mM CaCl 2 and plated at a concentration of 5 x 105/ml in 24-well culture plates that had been previously coated by treatment with FCS overnight. The cells were incubated at 37 C for 2 days to allow them to attach. Two types of experiment were then performed on the cells: Exp 1) Cells were cultured for a further 2 days in 0.5, 1.0 or 2.5 mM CaCl2, after which time they were harvested for RNA analysis. Exp 2) Cells were cultured for a further 2 days, in the absence or presence of 10-7 M 1,25(OH)2D3 (1 mM CaCl2 was used throughout), after which cells were harvested for RNA analysis.

Animals and experimental procedures
Normal male Sprague Dawley rats (Charles River Laboratories, Inc., St. Constant, Québec, Canada), weighing 180–200 g when received, were fed a standard rodent chow (Ralston Purina Co., LaSalle, Québec, Canada) containing 1.01% calcium, 0.74% phosphorus, and 3.3 IU vitamin D3/g. All animal experiments were carried out in compliance with, and were approved by, the institutional Animal Care and Use Committee. The following experiments were done: Exp 1) Rats were injected ip, at 48 and 24 h before death, with either vehicle (propylene glycol, 0.2 ml/100 g BW; or 1,25(OH)2D3, 50 or 250 pmol/100 g BW). Exp 2) Rats were injected ip, at 6 h before death, with calcium gluconate lactate 10% (2 ml) or PBS (2 ml). Exp 3) Rats were injected ip, at 6 h before death, with 5 µg synthetic salmon calcitonin (1 ml; Rhone-Poulence Rorer Canada Inc., Montréal, Québec, Canada) or PBS (1 ml). Exp 4) Five weeks before death, uremia was induced in rats by a one-stage, 5/6 nephrectomy procedure under pentobarbital anesthesia (60 mg/kg, ip). Control animals underwent a sham operation, which involved exposure of the kidneys and subsequent closure of the two separate flank incisions. After operation, rats were maintained on a diet containing 0.6% calcium, 1.4% phosphorus, TD.94238 (Harlan Teklad, Madison, WI). In all experiments, the rats were anesthetized with pentobarbital, a blood sample was collected by cardiac puncture, and the parathyroid glands were microdissected. Serum analyses were made as described previously (40).

Northern blot analysis
RNA was extracted from bovine parathyroid cells and rat parathyroid glands, and Northern blot analysis was carried out as described previously (38, 40). The hybridization probes used were as follows: 1) a human furin cDNA (28); 2) a rat PC7 cDNA (19); 3) a bovine PTH cDNA (41); 4) a rat PTH cDNA, kindly provided by Dr. Gerhard Heinrich, Boston, MA (42); and 5) a synthetic oligonucleotide complementary to the 3' end of rat 28S ribosomal RNA (40, 43). cDNA inserts were labeled with [32P]deoxycytosine triphosphate by the random primer method, and the oligonucleotide was labeled with [32P]ATP using T4 polynucleotide kinase.

RT-PCR
Five-microgram RNA samples were reverse transcribed with recombinant superscript II RNase H (Life Technologies, Inc., Gaithersburg, MD) using oligo(dT) 15–18 (Amersham Pharmacia Biotech, Inc., Baie d’Urfé, Québec, Canada) in a total vol of 20 µl. Four microliters of the RT mixture were subjected to standard PCR procedures. Primer pairs used are shown in Table 1Go. The PCR mixture contained 2 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM of each deoxynucleotide triphosphate, 50 pmol of forward and reverse primers, and 2.5 U Taq polymerase (Gibco BRL) in 100 µl. Thirty-five cycles (94 C, 40 sec; 57 C, 30 sec; 72 C, 45 sec) were performed with a programmable thermocycler (GeneAmp PCR System 9600, PE Applied Biosystems, Foster City, CA). Control RT-PCR reactions without RT were included to detect contamination of the RNA samples by genomic DNA. Aliquots were taken after 17, 20, 23, 26, 29, and 32 cycles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and after 20, 23, 26, 29, 32, and 35 cycles for the convertases and PTH; and 5 µl of each were electrophoresed through ethidium bromide-stained agarose gels, which were Southern-blotted and hybridized with the PCR product-specific 32P-labeled primers listed in Table 1Go. After washing and exposure to x-ray film, signal intensities were assessed using an LKB Ultroscan XL densitometer (LKB, Baie d’Urfé, Québec, Canada). All intensity values were corrected for the GAPDH signal, and a comparison of experimental vs. control values was made.


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  		Table 1. Sequences of the RT-PCR primers (F, forward; R, reverse) and internal hybridization probes (P) used

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Western blot analysis of ProPTH and PTH
Two different antisera were used for immunodetection of PTH moieties in the VV-infected GH4C1 cells. As shown in Fig. 1AGo, the hproPTH (-6–+7) antiserum recognized a polypeptide of electrophoretic mobility identical to that of recombinant hProPTH expressed in E. coli. It was selective for the proPTH precursor but did recognize the processed polypeptide, either as an E. coli recombinant hPTH (1–84) or an hPTH fraction from medium of the GH4C1 cells infected with VV:hPTH, albeit with much lower efficacy. The other antiserum used, which was raised against hPTH (1–84), was fully capable of recognizing both hproPTH and hPTH (Fig. 1BGo) and was used for the studies described in the subsequent section. These data demonstrate the utility of the electrophoretic system chosen in appropriately separating the proPTH and PTH moieties.



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  		Figure 1. Immunodetection of hproPTH and hPTH. Recombinant (rec) hproPTH and hPTH from E. coli extracts, and hproPTH and hPTH purified from VV:PTH-infected GH4C1 cells, as described in Materials and Methods, were electrophoresed through 20% polyacrylamide-SDS gels, run in tricine buffers. After electrotransfer to polyvinylidene difluoride membranes, specific polypeptide bands were detected by incubation with either a rabbit antiserum raised against hproPTH(-6-+7) (A) or a goat antiserum raised against hPTH (1–84) (B), and visualized, in each case, by chemiluminescence. Whereas the anti-hproPTH(-6-+7) antiserum only recognized proPTH, both proPTH and PTH were recognized by the hPTH (1–84) antiserum.

 
Coexpression of ProPTH and PCs in GH4C1 cells, effect of furin and PC7 on ProPTH processing
The resolution of immunoreactive PTH moieties from both GH4C1 cell and medium extracts was readily accomplished by RP-HPLC (see Figs. 2Go and 3Go). For the cell extract chromatography fractions, both proPTH and PTH were identified by Western blot analysis of chromatographic fractions (Fig. 3aGo), whereas for the medium extract, only PTH was found; no proPTH was detectable in the medium. As shown in Fig. 4Go, the immunoreactive species were definitively assigned as either proPTH and PTH (cells) or PTH (medium) by MALDI-TOF/MS. Therefore, further purification of the proPTH- and PTH-containing fractions (Fig. 4Go, A and B) from RP-HPLC of cell extracts was not necessary. For the medium, a single peak of immunopositive UV-absorbing material (Fig. 3bGo) was positively identified as PTH by the mass spectrometric analysis (Fig. 4CGo).



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  		Figure 2. RP-HPLC of cell extract (A) and medium (B) derived from culture of GH4C1 cells infected with VV:hPTH and VV:rdyn (control). In each instance, the column was eluted with a linear gradient of aqueous acetonitrile containing 0.1% (vol/vol) CF3COOH throughout. Column eluates were monitored for UV absorbance at 210 nm (continuous line), and column fractions were assayed for PTH immunoreactive moieties by Western blot analysis, which is shown in Fig. 3Go. Note that, for the elution profile of the medium sample (B), a peak of PTH immunoreactivity coincided with a single peak of UV-absorbing material, which was confirmed as hPTH-(1–84) using mass spectrometric analysis (see Figs. 3bGo and 4Go).

 


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  		Figure 3. Immunodetection of hproPTH and hPTH in RP-HPLC column fractions of cell extract (a) and medium (b) of GH4C1 cells infected with VV:hPTH and VV:rdyn (control). Insets a and b are expanded from boxed areas of absorbancy profiles of Fig. 2Go. Column fractions were subjected to Western blot analysis as described in Materials and Methods using the hPTH (1–84) antiserum to stain both hproPTH and hPTH.

 


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  		Figure 4. MALDI-TOF/MS analysis of RP-HPLC column fractions of cell extract and medium of GH4C1 cells infected with VV:hPTH and VV:rdyn (control). A, Fraction 20 (see Fig. 3aGo); B, fraction 23 (see Fig. 3aGo); C, fraction 23 (see Fig. 3bGo). The spectrograms show the determined masses for all peptides in the range 5,000–11,000 kDa found in each column fraction. Mass signals of 10149 (A and B) correspond to those expected for hproPTH, whereas mass signals of 9429 (B) and 9428 (C) correspond to those expected for hPTH (see Materials and Methods for details).

 
The relative amount of processing of proPTH under each condition was assessed from the ratio of PTH to proPTH, determined by densitometric scanning of Western blot autoradiographs (see Fig. 5Go). As shown in Fig. 5aGo, some processing by the endogenous convertases was evident in the control VV:rdyn-coinfected cells, with the ratio of PTH to proPTH being 0.07 (and set to 100% for comparative purposes). However, the ratio of PTH to proPTH was markedly increased, 484% that of the control, in cells coinfected with VV:hfurin (Fig. 5bGo). It was also increased, to 378% that of control, in cells coinfected with VV:rPC7 (Fig. 5cGo) and 356% that of control in cells coinfected with VV:5'KrPC7 (Fig. 5dGo). Therefore, in the latter case, modification of the native translation start site sequence of PC7 to a consensus Kozak start site sequence did not produce any increase in production of active protein.



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  		Figure 5. Immunodetection of hproPTH and hPTH in RP-HPLC column fractions of extracts of GH4C1 cells infected with VV:hPTH and VV:rdyn (control) (a), or VV:hfurin (b), or VV:rPC7 (c), or VV:5'KPC7 (d). Western blot analysis of column fractions was carried out as described in Materials and Methods using an antiserum that recognizes both proPTH and PTH. The PTH/proPTH ratios (see text) were determined from the relative densitometric intensities of the autoradiographic images.

 
Only PTH was released into the medium, and the relative amount secreted was similar under all conditions, despite marked differences in the efficiency of the intracellular conversion of proPTH to PTH noted above. This is exemplified by the data of Fig. 6Go, in which Western blot analysis of media samples from all four coinfection conditions are directly compared on a single blot. Amounts of immunoreactive PTH released under each condition were semiquantitated by densitometric analysis, and the values for the control, VV:rdyn, were set at 100%. For the other conditions, the values were as follows: VV:hfurin, 105%; VV:rPC7, 126%; and VV:5'KrPC7, 118%. It is likely that, in control cells overexpressing PTH, the available granules are already being maximally used; and when PTH production is increased by exogenous expression of a particular PC [previously furin (17), in the present study, PC7], the secretory apparatus is unable to increase its capacity further.



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  		Figure 6. Immunodetection of hPTH in RP-HPLC column fractions (no. 22, 23, and 24) of media of GH4C1 cells infected with VV:hPTH and VV:rdyn (control), or VV:hfurin, or VV:rPC7, or VV:5'KrPC7. Western blot analysis of column fractions was carried out as described in Materials and Methods using an antiserum that recognizes both proPTH and PTH. No proPTH was released into the conditioned medium. The relative amount of PTH released under each condition was determined from the relative densitometric intensities of the autoradiographic images.

 
Effect of calcium on furin mRNA levels in cultured bovine parathyroid cells
By Northern blot analysis, the furin cDNA probe identified an mRNA species of 4.7 kb (Fig. 7Go; left panel), and the PC7 cDNA probe identified an mRNA transcript of 4.2 kb (data not shown). In contrast to the reduction in PTH mRNA levels in response to an elevated medium calcium concentration, there was no modulation of the furin mRNA levels (Fig. 7Go) or PC7 mRNA levels. In parathyroid cells exposed to 10-7 M 1,25(OH)2D3 for 2 days, although there was a marked reduction in PTH mRNA levels, there was no change in the furin or PC7 mRNA levels (data not shown). The results are representative of those obtained in three independent experiments.



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  		Figure 7. Left-hand panel: Northern blot analysis of total RNA from bovine parathyroid cells cultured in low (0.4 mM) or high (2.5 mM) calcium concentration. Blots were hybridized with furin (A) and PTH (B) probes as described in Materials and Methods. C, Relative RNA quantity and quality were monitored by ethidium bromide staining. Right-hand panel: Parathyroid furin mRNA levels are not modulated by increased 1,25(OH)2D3. Northern blot analysis was conducted on parathyroid gland RNA from rats injected with either vehicle or 250 pmol 1,25(OH)2D3/100 g, at 48 and 24 h before death, as described in Materials and Methods. Although PTH mRNA levels were markedly reduced by 1,25(OH)2D3 administration, there was no change in parathyroid furin mRNA expression.

 
Effect of 1,25(OH)2D3 on rat parathyroid gland furin mRNA levels in vivo
Administration of 1,25(OH)2D3 (250 pmol/100 g) markedly decreased PTH levels in the parathyroid glands of normal rats, compared with vehicle-injected controls (Fig. 7Go; right panel). However, parathyroid gland furin (Fig. 7Go) and PC7 (data not shown) mRNA levels were unchanged in response to 1,25(OH)2D3 administration. The results are representative of those obtained in three independent experiments.

PC7 mRNA is expressed in the parathyroid
Expression in the parathyroid of PC7 mRNA and that of other proconvertases was examined by semiquantitative RT-PCR analysis (Fig. 8Go). This demonstrated that PC7 mRNA was present in parathyroid, in addition to confirming the presence of furin mRNA. The lack of PC1 mRNA expression in parathyroid was also confirmed, although abundant expression of this PC was demonstrated in the AtT20 mouse pituitary corticotroph cell line, as documented previously (44).



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  		Figure 8. Expression of PC7 and furin mRNA in rat parathyroid. RT-PCR was performed on RNA isolated from parathyroid glands of normal rats, as well as from neuroendocrine cell lines GH4C1 (positive control for furin, and PC7) and AtT20 (positive control for PC1), as described in Materials and Methods. Aliquots of PCR reactions were taken after 20, 23, 26, 29, 32, and 35 cycles and electrophoresed through ethidium bromide-stained gels.

 
Parathyroid gland PC mRNA levels are unaltered in response to changes in circulating calcium and 1,25(OH)2D3 concentrations
Serum calcium and 1,25(OH)2D3 levels were manipulated in vivo in rats by four different experimental procedures; and expression of parathyroid gland PTH, furin, and PC7 mRNA levels was assessed by semiquantitative RT-PCR analysis. The results are summarized in Fig. 9Go and Table 2Go. Whereas injection of 1,25(OH)2D3 clearly reduced PTH mRNA levels, there was no significant alteration in parathyroid gland furin and PC7 mRNA levels (Fig. 9Go). A summary of this data, as well as that from experiments in which serum calcium levels were increased, decreased (by calcitonin administration), or 1,25(OH)2D3 levels decreased by 5/6 nephrectomy, is shown in Table 2Go. Whereas the expected changes in PTH mRNA were documented in each case, there was no significant change in furin or PC7 mRNA levels.



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  		Figure 9. Parathyroid gland proconvertase mRNA levels in rats with increased circulating 1,25(OH)2D3 levels. RT-PCR of parathyroid RNA from rats, injected either with vehicle (control) or 50 pmol of the active vitamin D metabolite [1,25(OH)2D3], was carried out as described in Materials and Methods. Aliquots of PCR reactions taken after 20, 23, 26, 29, 32, and 35 cycles for PTH, and the convertases, or 17, 20, 23, 26, 29, and 32 cycles for GAPDH were electrophoresed through ethidium bromide-stained agarose gels. Densitometric analysis of Southern blots probed with internal sequence oligonucleotides showed that, whereas PTH mRNA was reduced by 60%, the convertase mRNAs remained unchanged in the glands of 1,25(OH)2D3-injected rats, relative to controls.

 

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  		Table 2. Rat parathyroid furin, PC7, and PTH mRNA levels in response to changes in serum 1,25(OH)2D3 and calcium

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several mammalian subtilisin-like serine endoproteases, having distinct or overlapping cleavage specificities, have been identified that process proteins by cleaving carboxyl-terminal to pairs of basic residues (for review, see Ref. 44). The most well-characterized members of this enzyme family are furin, PC1, and PC2. Furin is ubiquitously expressed, has a neutral pH optimum, contains a transmembrane domain, and functions in the trans-Golgi network; whereas PC1 and PC2 are localized exclusively in neuroendocrine cells, have more acidic pH optima, and act predominantly within secretory granules. In the parathyroid chief cell, proPTH is cleaved to PTH in the trans-Golgi network (4), rather than in secretory granules, which is consistent with processing of proPTH by furin or a furin-like enzyme rather than PC1 or PC2. Data in support of this hypothesis came with our demonstration that when hproPTH was coexpressed with either furin, PC1, or PC2 (in both a constitutively secreting cell-line or a neuroendocrine cell-line having a regulated secretory pathway), the most efficient processing was obtained with furin (17). Moreover, whereas furin is expressed in the parathyroid cell, PC1 and PC2 are not (17, 45).

The most recently identified member of the PC family is PC7 (19) [also known as LPC (20), PC8 (21), or SPC7 (22)], which (like furin) is membrane-anchored. An analysis, using partially-purified recombinant rPC7 and fluorogenic peptidyl substrates and synthetic peptides spanning known proprotein cleavage sites, showed the pH optima, Ca2+ requirement, and sequence specification of PC7 and furin to be broadly similar (23). In fact, to date, no precursor that is PC7-specific and is not also well-cleaved by furin has been identified. In the present coinfection studies, we showed that PC7, as well as furin, can appropriately cleave proPTH in cells having a regulated secretory pathway. Colocalization studies showed that PC7 is expressed in the parathyroid gland. Therefore, PC7 (like furin) has the potential to be involved in the physiological processing of proPTH to PTH.

Regulation of PC expression has been observed, and the direction of the changes often parallels that of the substrate of the convertase. For example, proTGFß is a furin substrate, and furin is up-regulated by cytokines (such as TGFß) in synovial and fibroblastic cells (46). During embryogenesis in the rat, furin mRNA becomes expressed in heart and liver and other tissues (47) coincident with expression of its substrates proTGFß (48) and proinsulin-like growth factor I (49). The furin gene promoter (50) contains potential cytokine-related responsive elements such as AP-1, and it has been shown that phorbol esters can increase furin gene expression in lymphocytes (51). In the rat pancreatic islet, where PC1 and PC2 have been implicated in the processing of proinsulin to insulin, glucose (which is the major stimulator of insulin biosynthesis and secretion) also up-regulates proinsulin processing. Glucose rapidly increases PC1 synthesis (52) by a posttranscriptional mechanism and may also similarly regulate PC2 biosynthesis (53).

In contrast to the glucose regulation of proinsulin processing in the pancreatic islet, earlier studies on conversion of metabolically-labeled proPTH to PTH in bovine parathyroid slices (54, 55) and parathyroid cells (56), in the short-term, found no evidence for regulation of proPTH processing by extracellular calcium, the predominant regulator of PTH gene expression and PTH secretion. Rather, there seemed to be a blood calcium-controlled degradative pathway that regulated the relative amount of intact (and therefore biologically-active) PTH available to be secreted (55). Under normal-to-high calcium conditions, little PTH was stored but rather was degraded to biologically-inactive fragments by an unknown mechanism (54, 57). It seems that enzymes of the PC type are not involved in a major way in this process (17), and the present study confirms this for PC7. It is now appreciated, however, that in vitro parathyroid cell cultures may be an unsatisfactory model to study some calcium-mediated events because the expression of the calcium-sensing receptor is often decreased in such cells (58, 59). In addition, the effects of the hormonally-active metabolite of vitamin D, 1,25(OH)2D3, on proPTH processing have never been assessed. Therefore, in the present study, we examined the potential modulation of proconvertase levels by extracellular fluid calcium and 1,25(OH)2D3 in vitro and in vivo. In bovine parathyroid cells cultured in low or high calcium for 2 days, PTH mRNA levels were modulated as expected, whereas no changes in furin mRNA levels were observed. Additionally, alteration of serum calcium and 1,25(OH)2D3 levels in vivo, which were achieved by several different protocols in the rat, while producing the anticipated changes in PTH mRNA levels, had no effect on parathyroid furin or PC7 mRNA levels. Thus, the results of our studies, examining the potential for regulation of the processing of proPTH to PTH by parathyroid convertases, confirm and extend the notion derived from earlier in vitro experiments that this step is not regulated by calcium. Furthermore, we also show that alterations in 1,25(OH)2D3, the other major regulator of parathyroid gland function, are not associated with changes in parathyroid convertase mRNA levels. Because regulation of PTH is not at the PC level, attention should now focus on understanding degradative mechanisms.


   Acknowledgments
 
We thank Susan James and Diane Savaria for their help with various aspects of this study, and Carmen Ferrara-Wilson and Pamela Kirk for preparation of the manuscript.


   Footnotes
 
1 This work was supported, in part, by Medical Research Council of Canada Grants MT-9315, MT-15057, MT-6733, and PG-11474. Back

2 Recipient of a studentship from the Medical Research Council of Canada. Back

Received January 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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